There are more than 60 serologically related insect-borne flaviviruses, including important human pathogens, such as the mosquito-borne yellow fever virus, Japanese encephalitis virus, and dengue viruses and the tick-borne encephalitis viruses. Among them, the four dengue virus serotypes (DEN1-4) are most important in terms of human morbidity which is estimated to involve millions of individuals every year. For this reason, the World Health Organization has assigned a high priority to the development of a dengue virus vaccine. Because of their importance in human disease, we investigated the molecular biology of the dengue viruses, focusing initially on genome organization and the mechanisms of gene expression and viral replication. It was thought that this information might allow us to gain a better understanding of dengue virus pathogenesis and eventually, to design molecular strategies for the prevention of dengue virus disease. DEN4 RNA genome, approximately 11 thousand nucleotides (nt) in size, codes for a long polyprotein that is cleaved to yield 10 viral proteins that include the three virion structural proteins, i.e., the capsid protein (C), the precursor membrane protein (PreM), and the envelope protein (E) and seven nonstructural proteins (NS). The DEN4 genome contains a 5' non-coding (NC) sequence and a 3' NC sequence, each of which is predicted to form a stable secondary structure that is thought to play a role during viral replication. After considerable effort, we successfully constructed a full-length DEN4 cDNA clone that yielded RNA transcripts that were infectious when transfected into susceptible cells in culture. A series of deletions varying in length were introduced into the 5' or 3' NC sequence region of the DEN4 cDNA clone; most of the deletion mutants proved to be viable. We showed that most viable 3' deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared to the wild type virus. Most 3' NC deletion mutants, similar to 5' NC deletion mutants constructed earlier, also replicated less efficiently and attained a lower titer in LLC-MK2 cells than the wild type virus. Recovery and identification of viable mutants that contained a deletion in the 3' NC region or 5'NC region and that were restricted in replication in simian LLC-MK2 cells, allowed us to prepare a menu of candidate attenuating mutations to be considered for use in a live dengue virus vaccine.